The present invention relates to a chimeric virus vaccine against tick-borne encephalitis virus (TBEV) and its other virulent relatives. More specifically, the invention relates to a chimeric virus comprising the Langat (LGT) virus preM and E structural protein genes linked to the non-structural protein genes of a mosquito-borne flavivirus.
The Flaviviridae family encompasses more than sixty antigenically related, positive strand RNA viruses within the arthropod-borne flavivirus genus, many of which are important human pathogens (Monath et al., Flaviviruses, in Virology, B. N. Fields et al., Eds., Raven Press, New York, pp. 961-1035, 1996). These include the mosquito-borne yellow fever virus, Japanese encephalitis virus, dengue viruses (DEN) and the tick-borne encephalitis viruses (TBEV), the latter being endemic in most European countries, Russia, India and North China. TBEV is transmitted exclusively by ticks and can be divided into two serologically distinguishable subtypes: the Eastern subtype (prototype strain Sofin), prevalent in Siberian and Far Eastern regions of Russia, and the Western subtype (prototype strain Neudorfl), common in eastern and central Europe. TBEV causes a serious encephalitic illness with a mortality rate ranging from 1 to 30%.
Flaviviruses share the same genome organization: 5xe2x80x2-C-preM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3xe2x80x2 in which the first three genes code the capsid (C), premembrane (preM) and envelope (E) proteins, while the remainder of the genes encode nonstructural proteins. Homology between mosquito-borne and tick-borne flaviviruses is relatively low (Chambers et al., Annu. Rev. Microbiol. 44:649-688, 1990; Pletnev et al., Virology 174:250-263, 1990). However, homology among mosquito-borne flaviviruses or among tick-borne flaviviruses is relatively high (Iacoco-Connors et al., Virology 188:875-880, 1992).
Four serotypes of dengue virus are known (type 1 to type 4) which are distinguishable by plaque reduction neutralization using serotype-specific monoclonal antibodies and by less specific tests using polyclonal sera (Bancroft et al., Pan Am. Hlth. Org. Sci. Publ. 375:175-178, 1979; Henchal et al., Am. J. Trop. Med. Hyg. 31:548-555, 1982). The four dengue serotypes share a common genome organization. The complete nucleotide sequences have been determined for dengue virus types 3 and 4 and several strains of type 2 virus including the mouse-neurovirulent New Guinea C mutant (Mackow et al., Virology 159:217-228, 1987; Zhao et al., Virology 155:77-88, 1986; Osatomi et al., Virology 176:643-647, 1990; Irie et al., Gene 75:197-211, 1989; Mason et al., Virology 161:262-267, 1987; Hahn et al., Virology 162:167-180, 1988).
Despite the considerable evolutionary distance between DEN and TBEV, a viable chimeric flavivirus was constructed which contained the C-preM-E or preM-E structural protein genes of a virulent Far Eastern Russian TBEV with the remaining nonstructural protein genes and 5xe2x80x2- and 3xe2x80x2-noncoding sequences derived from DEN4 [TBEV(CME)/DEN4 and TBEV(ME)/DEN4, respectively] (Pletnev et al., Proc. Natl. Acad. Sci. U.S.A. 89:10532-10536, 1992).
TBEV(ME)/DEN4 retained the neurovirulence in mice of its TBEV parent from which its preM and E genes were derived, but it lacked the peripheral neurovirulence of TBEV, i.e. the ability to spread from a peripheral site to the central nervous system (CNS) and cause fatal encephalitis. However, mice previously inoculated with the chimeric virus by a peripheral route were completely resistant to subsequent intraperitoneal challenge with a lethal dose of the highly virulent TBEV. Neurovirulence of this chimera was significantly reduced by a single mutation introduced into its preM, E or nonstructural protein 1 (NS1) viral protein (Pletnev et al., J. Virol. 67:4956-4963, 1993). These amino acid substitutions also caused a restriction in viral replication in tissue cultures of both simian and mosquito cells. Nonetheless, parenteral inoculation of these further attenuated chimeric mutants induced complete resistance in mice to fatal encephalitis caused by intracerebral inoculation of the neurovirulent TBEV(ME)/DEN4 chimera.
Langat (LGT) virus is the least virulent of all TBEV-complex flaviviruses, but is closely related antigenically to the highly virulent Far Eastern TBEV (Calisher et al., J. Gen. Virol. 70:37-43, 1989; DeMadrid et al., J. Gen. Virol. 23:91-96, 1974; Iacoco-Connors et al., Virus Res. 43:125-136, 1996) and has a high level of sequence homology thereto (Iacoco-Connors et al., Virology 188:875-880, 1992; Mandl et al., Virology 185:891-895, 1991; Shamanin et al., J. Gen. Virol. 71:1505-1515, 1990). LGT virus was tested as an experimental live vaccine against TBEV during the early 1970s (Ilenko et al., Bull. Wld. Hlth. Org. 39:425-431, 1968; Mayer et al., Acta. Virol. 19:229-236, 1975; Price et al., Bull. Wld. Hlth. Org. 42:89-94, 1970). Several LGT strains which were attenuated for mice and monkeys were isolated and tested in 800,000 adults; however, clinical trials were discontinued when vaccination with one of the most attenuated vaccine candidates, Yelantsev virus, was associated with a very low frequency of encephalitis, i.e. one case per 20,000 vaccinations (Mandl et al., supra.)
Currently, an experimental TBEV vaccine produced by formalin inactivation of TBEV is available; however, this vaccine has several limitations. For example, the vaccine is not strongly immunogenic, therefore repeated vaccinations are required to generate a protective immune response. Even when antibody responses to the vaccine are present, the vaccine fails to provide protective responses to the virus in 20% of the population. Therefore, there remains the need for a safe, more effective vaccine against TBEV. The present invention provides such a vaccine.
One embodiment of the present invention is a viable chimeric recombinant flavivirus, comprising a first region of nucleic acid operably encoding preM and E structural proteins of Langat virus operably linked to a second region of nucleic acid operably encoding non-structural proteins of a mosquito-borne flavivirus. Preferably, the Langat virus is the Langat wild type virus strain TP21 or its further attenuated mutant, Langat strain E5. Advantageously, the mosquito-borne flavivirus is a dengue virus. In one aspect of this preferred embodiment, the dengue virus is type 4. Alternatively, the mosquito-borne flavivirus is yellow fever virus. According to another aspect of this preferred embodiment, the first region of nucleic acid also operably encodes capsid protein from the mosquito-borne flavivirus or from Langat virus. In yet another aspect of this preferred embodiment, the recombinant flavivirus further comprising at least one mutation. Preferably, the recombinant flavivirus is incorporated within an expression vector. Advantageously, the expression vector is a plasmid.
The present invention also provides a host cell stably transformed with the recombinant flavivirus described above in a manner allowing expression of said DNA construct. Preferably, the host cell is prokaryotic. In another aspect of this preferred embodiment, the tick-borne encephalitis virus is selected from the group consisting of the Eastern, Western, Omsk hemorrhagic fever, louping ill, Kyasanur forest disease, Negishi, or Powassan viruses.
Another embodiment of the present invention is a vaccine against tick-borne encephalitis virus, comprising the chimeric recombinant flavivirus described above in a pharmaceutically acceptable carrier. Another embodiment is the vaccination of milk producing mammals against tick-borne encephalitis virus infection. Still another embodiment of the present invention encompasses an immunogenic composition comprising the chimeric recombinant flavivirus described above in a pharmaceutically acceptable carrier.
The present invention also provides a method of preventing TBEV infection in a mammal, comprising the step of administering to the mammal an effective TBEV-preventing amount of a chimeric recombinant flavivirus, the chimeric flavivirus comprising a first region of nucleic acid operably encoding C, preM and E structural proteins of Langat virus, or C protein of the mosquito-borne flavivirus plus preM and E structural proteins of Langat virus, operably linked to a second region of nucleic acid operably encoding non-structural proteins of a mosquito-borne flavivirus, in a pharmaceutically acceptable carrier. Preferably, the mammal is a human. Advantageously, the administering step is intranasal, intradermal, subcutaneous, intramuscular, or intravenous. In one aspect of this preferred embodiment, the effective TBEV-preventing amount is between about 1 xcexcg and 1,000 xcexcg. The method may further comprise administering one or more booster injections of the chimeric flavivirus.
The present invention also contemplates a method of stimulating an immune response directed against the chimeric recombinant flaviviruses discussed above, in a mammal, comprising the step of administering to the mammal an effective TBEV-preventing amount of a chimeric recombinant flavivirus, the chimeric flavivirus comprising a first region of nucleic acid operably encoding C, preM and E structural proteins of Langat virus, or C protein of the mosquito-borne flavivirus plus preM and E structural proteins of Langat virus, operably linked to a second region of nucleic acid operably encoding non-structural proteins of a mosquito-borne flavivirus, in a pharmaceutically acceptable carrier.